This proposal addresses one of the longest standing challenges in biology: how to enhance wound healing and regeneration in mammals. Regeneration has been studied for centuries, but many questions remain unanswered. Why do mammals have a limited ability to regenerate compared to other vertebrates, such as salamanders and fish? What are the molecular mechanisms that control regeneration? Is enhancing regeneration possible in mammals? If mammals have the potential to regenerate, what are the limiting factors? Can regeneration be activated and enhanced pharmacologically? We propose that wound repair and regeneration can be enhanced by reducing the production of heparan sulfate, a type of glycosaminoglycan expressed by all animal cells. Heparan sulfate is often considered a cofactor, because it binds to many other cell surface, secreted and extracellular matrix proteins, thereby modulating their activities. A complete loss of heparan sulfate is not compatible with life, but animals and humans tolerate variation in heparan sulfate content and structure, typically resulting in loss-of-function phenotypes. Here, we propose that the loss of heparan sulfate results in a gain-of-function, specifically in enhanced cutaneous wound repair and digit regeneration. The suggestion that creating a macromolecular deficiency in a mammal can enhance repair and regeneration is paradigm-shifting in a field mostly concerned with activation of latent pathways and delivery of growth and morphogenic factors. In Aim 1, using heparan sulfate deficient mice, we will examine six models of wound repair and regeneration that allow us to test the scale of tissue regenerative potential from epidermis and dermis in excisional skin wounds, to hair and fat regeneration in large wounds, cartilage regeneration in ear punches, and finally to regeneration of complex multiple tissue structures in digit amputations. In each model we will characterize the rate and extent of wound healing and regeneration. Based on our preliminary experiments, glycoside treatment of mice also phenocopies the enhanced wound healing in the unsplinted full thickness dorsal skin excisional model. In Aim 2, we will optimize glycoside treatment by varying the structure of the glycoside, analyzing dose response profiles and determining the timing of treatment relative to wounding. In the R33 Phase we will examine the impact of altering glycosaminoglycan assembly in disease-induced wound models using the diabetic db/db mice as a model. db/db mice will be crossed into a heparan sulfate deficient background and examined for regenerative ability in wounding models. Finally, we will treat wounded diabetic mice with glycosides to determine the therapeutic potential of heparan sulfate reduction in enhancing wound healing and regeneration.